South African Journal of Botany 87 (2013) 1–3

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South African Journal of Botany

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Antimicrobial activities of thouarsii Roemer and Schulte, a traditional medicinal from Madagascar

Vahinalahaja Razafintsalama a, Samira Sarter b,f, Lengo Mambu c, Ranjana Randrianarivo a, Thomas Petit d, Jean François Rajaonarison e, Christian Mertz f, Danielle Rakoto a, Victor Jeannoda a,⁎ a Department of Fundamental and Applied Biochemistry, Faculty of Sciences, University of Antananarivo, BP 906, Antananarivo 101, Madagascar b CIRAD, UMR QUALISUD, 101 Antananarivo, Madagascar c UMR 7245 CNRS-MNHN, Molécule de Communication et Adaptation des Micro-organismes, Muséum National d'Histoire Naturelle, 63 rue Buffon, 75005 Paris, France d Université de la Réunion, Laboratoire de Chimie Des Substances Naturelles et Des Sciences des Aliments (LCSNSA), 15 Avenue René Cassin, 97 715 Saint-Denis La Réunion, France e Institut Malgache de Recherche Appliquée (IMRA), BP 3833 Avarabohitra Itaosy, Antananarivo, Madagascar f CIRAD, UMR QUALISUD, F-34398 Montpellier, France article info abstract

Article history: The leaves of Dilobeia thouarsii (Roemer and Schulte), a that is endemic to Madagascar (), are Received 24 April 2012 used in traditional Malagasy medicine to treat bacterial skin infections and wounds. This study investigated Received in revised form 22 June 2012 the in vitro antibacterial activities of D. thouarsii leaf extracts and identified the bioactive compounds with Accepted 22 February 2013 the aim of providing a scientific basis for its use against skin diseases. Using broth microdilution method Available online 2 April 2013 for leaf crude extract and its compounds, we investigated inhibition of the growth of Bacillus cereus, fi Edited by J Van Staden Bacillus megaterium, Staphylococcus aureus, Enterococcus faecalis, Vibrio harveyi, Vibrio sheri, Salmonella Typhimurium, Salmonella antarctica, Escherichia coli, and Klebsiella pneumoniae. The two purified phenolic Keywords: compounds from leaf ethyl acetate extracts (1, 2) were found to be more active than the crude extract itself. Dilobeia thouarsii The structure of the two compounds was elucidated by NMR and mass spectrometry: compound 1 was Proteaceae identified as 4-aminophenol and compound 2 as 4-hydroxybenzaldehyde. A marked inhibitory effect Medicinal plant (MIC b 0.1 mg/ml) was found against S. aureus, which is a major agent in skin infections. We observed Antibacterial moderate activities (MIC values of between 0.1 and 0.5 mg/ml) for E. faecalis, Vibrio spp., and Bacillus spp. Neither Plant extract compound was active against Salmonella spp., E. coli and K. pneumoniae (MICs > 1 mg/ml). To conclude, the high Phenolic compound antimicrobial activity of D. thouarsii leaf extracts against S. aureus supports its traditional use to treat skin infections. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction In vitro assays have shown that phenolic compounds are often responsible for the antimicrobial activities of different plant extracts Traditional medicine is an important component of the health care (Shikanga et al., 2010; Tepe et al., 2005; Zampini et al., 2005). Several system in Madagascar and a large number of remain to be species belonging to the family Proteaceae, such as robusta, studied, including Dilobeia thouarsii, a tree that belongs to the family toru, avellana, elliptica, obtusifolia Proteaceae and is endemic to Madagascar (Boiteau, 1986). This or hirsuta contain phenolic compounds (Ahmed et al., 2000; species is widely distributed in the Central, Eastern, South-Eastern Chuang and Wu, 2007; Moure et al., 2001; Perry and Brennan, regions and in the high Matsiatra Fianarantsoa in Madagascar 1997; Simonsen et al., 2006; Verotta et al., 1999). In addition, species (Bosser and Rabevohitra, 1991) and is known by the common belonging to this family display antimicrobial activities against different names of Vivaona, Hazontavolo and Tavolohazo (Rabesa, 1986). In microorganisms. L. hirsuta, which is used in traditional medicine southern Madagascar, decoctions of the leaves and bark of D. thouarsii in Chile, is active against the pathogenic fungus Candida albicans are used for abortion, or as an anthelmintic, or a diuretic (Beaujard, (MIC = 8 μg/ml) (Simonsen et al., 2006). A phenolic glycoside ester 1988; Rabesa, 1986). Concerning the East coast of Madagascar isolated from the New Zeland tree T. toru is active against Pseudomonas (Mandraka region), our ethnobotanical investigations confirmed aeruginosa, Escherichia coli and Bacillus subtilis (Perry and Brennan, the use of the leaves in traditional medicine to treat bacterial skin 1997). A glycoside compound isolated from linearis × pinifolia, infections and wounds (Razafintsalama, 2012). a cross hybrid of P. pinifolia and P. linearis, displays antimicrobial activity against E. coli and Phytophthora cinnamoni (MacLeod et al., 1997). An ⁎ Corresponding author. Tel.: +261 32 02 402 20. extract made from leaves of Protea simplex,aplantusedinSouthAfrica E-mail address: [email protected] (V. Jeannoda). against human dysentery and diarrhea, provides good antimicrobial

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.02.171 2 V. Razafintsalama et al. / South African Journal of Botany 87 (2013) 1–3 activities against E. coli, Staphylococcus aureus, B. subtilis and C. albicans dissolved in sterile distilled water. The concentration of the resulting (Fawole et al., 2009). solutions was adjusted to 7 mg/ml. This was serially diluted twofold To the best of our knowledge, no report has been published on the to obtain concentration ranges of 0.027–7mg/ml.Next,100μl of each chemical composition and the biological activities of D. thouarsii concentration was added in a well (96-well microplate) containing (Bosser and Rabevohitra, 1991). In the present study, we investigated 95 μl of Zobell medium for vibrios (1 g/l yeast extract, 4 g/l peptone, the antibacterial activity of D. thouarsii and identified bioactive 30 g/l NaCl) or Mueller-Hinton broth for the other microorganisms compounds in order to provide a scientific basis for its traditional use, and 5 μl of inoculum (standardized at 1.5 × 106 cfu/ml by adjusting and to characterize the potential of this medicinal plant in Madagascar. the optical density to 0.125 at 600 nm). A positive control containing Bioassay fractionation enabled isolation of two phenolic compounds the bacterial culture without the extract and a negative control that were identified on the basis of spectroscopic data including 1D containing only the medium were also analyzed. The plates were NMR and mass spectrometry (MS). covered with sterilized aluminum foil, and then incubated for 24 h at 25 °C for Vibrio sp. and at 37 °C for the other strains. The assay was 2. Material and methods repeated three times. The MIC of each compound was defined as the lowest concentration that inhibited the microorganism growth. Bacterial 2.1. Plant material growth was visually evaluated based on the degree of turbidity (Kil et al., 2009). The leaves of D. thouarsii were harvested in Mandraka region, in the For the determination of MBC, 5 μl from each well not showing eastern part of Madagascar, 70 km from Antananarivo. Leaves were col- turbidity was placed on Mueller-Hinton agar and incubated at 37 °C lected in April 2008. The plant was identified by Dr. Rabarison Harison for 24 h. The lowest concentration at which no growth occurred on from the Botany Department of Antananarivo Faculty of Sciences. the agar plates after 24 h of incubation at 37 °C corresponded to the Reference specimens (HERB/DBEV/4708) were deposited in the MBC. herbarium of the same department of the University of Antananarivo.

2.2. Extraction of D. thouarsii leaves 3. Results and discussion

Plant materials were dried at room temperature and ground to a fine 3.1. Active compounds identified powder. The obtained powder (100 g) was extracted successively through a maceration process using 500 ml × 6 of solvents of increas- Compound 1 (Fig. 1) was isolated as an amorphous powder. ing polarity (hexane, ethyl acetate and methanol). Each combined HRESI-TOF performed in the negative mode exhibited a deprotonated extract was evaporated under reduced pressure to yield crude hexane molecular ion at m/z 108.0435 [M − H]− indicating a molecular extract (0.7 g), EtOAc extract (5 g), and MeOH extract (10 g), respec- formula of C6H7NO (calcd. 108.0447) requiring 4° of unsaturation. tively. Extracts were stored at room temperature until use. The 13C NMR spectrum revealed the presence of an oxygenated quaternary carbon at δ 151.2, another quaternary carbon at δ 117.4 2.3. Bioassay-guided extract and a methine carbon at δ 115.8. The 1H NMR spectrum of this small molecule displayed an intense Part of the ethyl acetate extract (1.5 g) was subjected to flash signal of four aromatic protons at δ 6.49. As the spectra were realized chromatography on a silica gel 60 (10–40 μ) column (CC) eluted in CD3OD, the three remaining protons not observed as suggested the with 0–100% gradient of EtOAc in hexane followed by MeOH in molecular formula are exchangeable protons. Comparison with RMN EtOAc. Fourteen 100 ml fractions were collected: Hex–EtOAc 80:20 data of the sample indicated that compound 1 was a 4-aminophenol (1–4), Hex–EtOAc 40:60 (5–8), Hex–EtOAc 20:80 (9–12), EtOAc (Sigma-Aldrich catalog). (13), and MeOH (14). On the basis of the analytic TLC, and according The molecular formula of compound 2 (Fig. 1) was deduced as to the antimicrobial assay, similar active fractions 5–8 (0.15 g) C7H6O2 from the deprotonated molecular ion peak at m/z 122.0368 were combined and rechromatographed on the same support using [M − H]− observed in the HRESI-MS compatible with four degrees the same solvent system. Fourteen new fractions were obtained, of unsaturation. Its 1H NMR spectrum showed the presence of an but only two displayed antibacterial activity. Each active fraction aldehyde proton at δ 9.73 and two doublets at δ 6.9 (2H, J = 8.4 Hz, was treated with 75% ethanol and concentrated to yield compounds H-3; H-5) and 7.8 (2H, J = 8.4 Hz, H-2; H-6). In comparison with 1, 1 (100 mg) and 2 (40 mg). The antimicrobial activity of these com- the difference of 13 uma suggested that the amino group was replaced pounds was evaluated on Gram-positive and Gram-negative bacteria.

2.4. Antimicrobial assays

2.4.1. Microorganism strains Four Gram-positive (Bacillus cereus LMG 6910, Bacillus megaterium NH CHO LMG 7127, S. aureus ATTC 25920, Enterococcus faecalis ATTC 29212) 2 and six Gram-negative bacteria (Vibrio harveyi ATCC 14126, Vibrio fisheri 4 4 ATCC 49387, Salmonella Typhimurium ATCC 14028, Salmonella antarctica LMG 3264, E. coli CCM 451, Klebsiella pneumoniae ATTC 13883) were used to study antibacterial activity. The bacteria were obtained from the collections of both the University of La Réunion (LCSNSA: Laboratoire 1 1 deChimiedesSubstancesNaturellesetdesSciencesdesaliments, Saint Pierre) and Cirad (Montpellier, France). OH OH

2.4.2. MIC and MBC determination 12 The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were evaluated using the microdilution Fig. 1. Structure of the two purified compounds 1 and 2 from the leaf ethyl acetate method described by Kuete et al. (2009). The samples were first extract of Dilobeia thouarsii. V. Razafintsalama et al. / South African Journal of Botany 87 (2013) 1–3 3 by an aldehyde group (Chen et al., 1999). Consequently, compound 2 4. Conclusion was identified as 4-hydroxybenzaldehyde. Its antimicrobial activity against S. aureus, a major agent in skin infections, provides a scientific basis for the traditional Malagasy use 3.2. Antibacterial activity of D. thouarsii (Roemer and Schulte) in the treatment of skin infec- tions. The two purified phenolic compounds from leaf ethyl acetate This is the first time that antimicrobial activity of D. thouarsii extracts extracts involved in this antimicrobial activity were 4-aminophenol has been reported. The two phenolic compounds determined from the and 4-hydroxybenzaldehyde. Consequently, leaf ethyl acetate extract leaf ethyl acetate extract (4-aminophenol and 4-hydroxybenzaldehyde) could be used in further investigations to identify the other molecules were more active against both Gram-positive and Gram-negative bacteria present in this plant. than the crude extract itself (Table 1). MIC and MBC values varied with the extracts and compounds tested. S. aureus was the most sensitive Acknowledgments strain. According to Oussou et al. (2008), the ratio observed for MBCs and MICs (MBC:MIC b 4) indicated that the bactericidal effect of the This work was financially supported by the project “Pôle d'Excellence compounds on the majority of strains tested could be expected. Globally, Régional” (AUF/ref/no. 2708PL708) funded by AUF (Agence Universitaire Gram-positive bacteria were more sensitive to these compounds than de la Francophonie). Gram-negative ones (Table 1). MIC values obtained with the two compounds for S. aureus were References lower than those of leaves and bark extracts of P. simplex (a Proteaceae from South Africa) which ranged between 0.147 and 0.780 mg/ml Ahmed, A.S., Nakamura, N., Meselhy, R.M., Makhboul, A.M., El-Emary, N., Hattoria, M., (Fawole et al., 2009). MIC values of our extracts were lower than 2000. Phenolic constituents from . Phytochemistry 53, 149–154. 0.1 mg/ml for S. aureus, which, according to Holetz et al. (2002), Beaujard, P., 1988. 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